Individual respiratory complexes of mitochondria are in dynamic equilibrium with higher order supercomplex organizations that compose the respirasome. Physiological and pathological perturbations in the mitochondrial cardiolipin (CL) pool directly affect and regulate this equilibrium. Aging, neurodegenerative diseases, heart failure, ischemia/reperfusion, cancer and Barth Syndrome are associated with abnormal CL pools. Saccharomyces cerevisiae mutants lacking CL display similar phenotypes to mammalian cells with reduced CL levels making yeast an excellent model system. We were the first to report that formation of the yeast tetrameric respiratory supercomplex (SC III2IV2) and cytochrome c (cyt c) channeling between complexes III (CIII, bc1 complex) and IV (CIV, cyt c oxidase) within the SC are directly dependent on CL. Using our recently acquired K2 Summit direct electron detector for single particle electron cryo-microscopy, we have significantly improved the quality of EM images. We achieved a higher resolution 3D density map of the tetrameric SC than we previously reported. We are now in a position to attain 3D density maps of SCs at an unprecedented sub- nanometer resolution. We propose an innovative combination of high resolution structural determinations, functional assays, genetic manipulation of yeast cells and novel lipid-dependent reconstitution studies to establish the molecular basis for CL-dependent formation and function of respiratory SCs. Aim 1: A) obtain a sub-nanometer resolution 3D density map for the tetrameric SC to establish the precise dimensions of the lipid- filled gaps and the interface between CIII and CIV; B) determine the location(s) of bound cyt c in the tetrameric SC to decipher how SC formation makes cyt c channeling possible; C) resolve the structure of the III2IV1 trimer to understand how structural differences with the tetramer results in CL-independent formation and lack of cyt c channeling in the trimer; D) perform structural studies of the SC tetramer lacking subunit Qcr6 of CIII coupled with lipid analysis (under Aim 2) to determine whether Qcr6p maintains the lipid-filled gap between CIII and CIV, which makes SC formation sensitive to CL levels. Aim 2: A) integrate structural with quantitative analysis of CL and other lipids present in the above SCs; B) determine the features of CL that support tetrameric SC formation and function using an innovative in vitro SC-reconstitution system employing structural analogs of CL; C) mimic pathological conditions resulting in CL pool alterations to understand how CL levels and CL oxidation affect SC structure and function. Aim 3: A) determine CIII and CIV surface exposed CL-binding sites potentially responsible for SC formation using a photoactivatible CL analog; B) use Molecular Dynamic Simulations to predict surface exposed CL-binding sites; C) employ site-directed mutagenesis at chemically modified and predicted CL-binding sites that lie at the CIII-CIV interface for verification of their involvement in SC formation. This innovative and integrated approach will establish the molecular mechanism of CL- dependent SC formation and function. In the future similar studies will be extended mammalian respirasomes.